Neuroplasticity is central to fundamental processes in the brain, including learning, and long-lasting changes in neural circuits that result from employing substances of abuse. In contrast to significant advances at the cellular/molecular level, our understanding of the functional organization and reorganization of brain circuits remains limited. Increasing in vivo evidence suggests that single cells remain plastic into and during adulthood. However, our knowledge of the functional properties of single cells is primarily descriptive. This limits our understanding of the underlying mechanisms involved in assembling and modifying neuronal tuning functions during plasticity. In order to understand how the properties of cells change during plasticity, it is imperative to record from a population of interconnected neurons in vivo. More than half of all synaptic contacts in the cortex arise from neurons within a -200 j.lm radius from the target cell. Importantly, connections between cells in the cerebral cortex are predominantly along cortical depth. Therefore, we need to monitor simultaneously the activity of all adjacent neurons in a cortical volume, and thus record in three dimensions. To date, no experimental tool exists that would allows us to do this. This project seeks to establish novel in vivo methods that will allow us to analyze neural circuits in three dimensions. For this purpose, we will advance two technologies to record from and manipulate circuits in the mammalian cortex: (1) Ultra-fast three-dimensional two-photon imaging, and (2) Optical manipulation of neural activity with single-cell and single-spike resolution using optogenetic tools. The proposed developments have become possible because of recent technical advances in ultra-fast multi-photon microscopy and light-activated ion channels. In short: Inertia-free nearinfrared laser scanning technology allows for in vivo fast structural and functional imaging as well as for high resolution optical stimulation. The proposed project will combine these key technologies to generate the infrastructure and the experimental skills to study the function and plasticity of cortical microcircuits and thus will help researchers to understand mechanisms of neuroplasticity in the intact brain.